Leishmania spp. are favorably considered as a universal carrier for vaccine delivery, since they have evolved not only an innate ability to infect antigen-presenting cells (APC) but also to reside exclusively in the antigen-processing phagolysosomes of macrophages. In addition, some species are relatively innocuous, causing human simple cutaneous leishmaniasis, which is known to heal spontaneously, resulting in life-long immunity. Immunization of human populations with live Leishmania (Leishmanization) has been indeed successful, but it still produces the skin disease, which is potentially debilitating and thus undesirable. It is possible to increase the safety margin of live Leishmania for vaccination by molecular genetic approaches to attenuate their pathogenicity or to construct suicidal mutants. These live vaccines have not been developed beyond the experimental stage, since they produced incomplete protection and/or persisted in animal models. We have recently developed a different type of transgenic Leishmania, which retains their natural ability of homing to the phagolysosomes of APC, but is inducible subsequently to accumulate uroporphyrin for selective photolysis therein. Timely induction of their self-destruction therein thus releases vaccines to the desirable site. Photodynamic vaccination of hamsters with these suicidal mutants was indeed found to simulate "Leishmanization", but without the skin disease, against visceral leishmaniasis. Moreover, the immunity was elicited without apparent persistence of the parasites and with long-term memory, as shown by its adoptive transfer to naive animals. Similar photodynamic vaccination of BALB/c mice was however found to protect them less completely against cutaneous leishmaniasis. The less robust outcome seen in this model is attributable to the escape of the mutants from externally administered inducers of Leishmania-specific uroporphyria and/or photolysis. We propose to improve the suicidal designs by rectifying the mutant deficiencies as follows: [1] Transfection of the porphyrinogenic mutants for phagolysosomal stage-specific expression of an additional enzyme to timely produce delta-aminolevulinate (ALA), thereby resulting in the self-induction of uroporphyria;and [2] Further genetic engineering of the mutants to express luciferase for phagolysosomal stage-specific generation of light to enhance their cytolysis after uroporphyrinogenesis. The relevant transgenes will be placed under the regulation of known 3'-UTR sequences for stage-specific expression as the mutants reach phagolysosomes for differentiation. Combination mutants of [1] and [2] using an ALA synthase plus a luciferase will be constructed in attempt to achieve their self-induction of uroporphyrinogenesis as well as luciferin-inducible emission of light. This design of automation is thus expected to render photolysis of the mutants systemic and timely, independent of the external inducers applied to the site of inoculation. Application of such suicidal mutants will increase both immunogenicity and safety margin, thereby enhancing their potential use as a universal vaccine platform for photodynamic vaccination. PUBLIC HEALTH RELEVANCE: The work proposed in this application is to develop, improve and evaluate inducible suicidal mutants of parasitic protozoa, Leishmania, which have the natural ability to enter and live in our immune cells specialized in accepting vaccines to make them effective. The outcome of the study will help us produce a universal carrier to deliver vaccines for preventing and treating infectious and non-infectious diseases.